Methods of typing hepatitis C virus and reagents for use therein

ABSTRACT

The claimed invention provides methods of detecting and typing HCV using type specific and type-cluster specific epitopes. The claimed invention also provides peptides having type specific and type-cluster specific epitopes.

This application is a continuation-in-part, continuation, division, ofapplication Ser. No. 08/060,400, filed May 10. 1993 abandoned.

FIELD OF THE INVENTION

This invention relates to typing hepatitis C viruses (HCV). Inparticular, this invention relates to a method of typing HCV using noveltype specific polypeptides.

BACKGROUND OF THE INVENTION

Viral hepatitis is known to be caused by five different viruses known ashepatitis A,B,C, D and E. HAV is an RNA virus and does not lead tolong-term clinical symptoms. HBV is a DNA virus. HDV is a dependentvirus that is unable to infect cells in the absence of HBV. HEV is awater-borne virus. HCV was first identified and characterized as a causeof non-A, non-B hepatitis (NANBH). Houghton et al., EPO Pub. No.388,232. This led to the disclosure of a number of general and specificpolypeptides useful as immunological reagents in identifying HCV. See,e. g., Choo et al. (1989) Science, 244:359-362; Kuo et al. (1989)Science, 244:362-364; and Houghton et al. (1991) Hepatology, 14:381-388.HCV is the major cause of blood transfusion-related hepatitis.

The prototype isolate of HCV was characterized in EP Publication Nos.318,216 and 388,232. As used herein, the term "HCV" includes newlyisolated NANBH viral species. The term "HCV-1" refers to the virusdescribed in the above-mentioned publications.

Since the initial identification of HCV, at least six different viraltypes have been identified and designated HCV-1 to HCV-6. Cha et al.(1992) Proc. Natl. Acad. Sci. USA, 89:7144-7148. Within these types arenumerous subtypes. The type of virus with which a patient is infectedmay affect the clinical prognosis and also response to varioustreatments. Yoshioka et al. (1992) Hepatology, 16:293-299. In light ofthe fact that the most serious clinical outcome of HCV infection ishepatocellular carcinoma, it would be useful to be able to determinewith which type or types of HCV a patient is infected.

The method currently in use to determine virus type is genotyping; thatis, isolation of viral RNA and determination of the sequence of varioussegments by polymerase chain reaction (PCR). Not only is this methodlaborious and time consuming but it is not suitable for use on samplesthat have been stored under conditions that do not allow forpreservation of RNA or samples from patients that do not have sufficientviral titer. It would be useful to have a method for typing HCV byimmunoanalysis or serotyping.

The current method for screening blood and diagnosing patients is animmunoassay. The immunoassay utilizes an antigen from HCV-1 whichcontains a sufficient number of common epitopes to detect antibodies toother types of HCV. The immunoassay does not distinguish betweeninfections by different types of HCV.

SUMMARY OF THE INVENTION

The present invention includes compositions and methods for typing ofHCVs by genotype and serotype. The compositions include type specificepitopes, type-cluster specific epitopes, nucleic acids encoding theepitopes for use as probes and nucleic acids complementary to theregions flanking those encoding the epitopes for use as primers.

One aspect of the invention is a method for typing HCV comprising thesteps of providing an antibody-containing sample from an individual;contacting the sample with a type specific epitope or type-clusterspecific epitopes under conditions which permit antigen-antibodybinding; and determining whether antibodies in the sample bind to theepitope.

Another aspect of the invention relates to a method for typing HCVcomprising the steps of providing an antibody-containing sample from anindividual; contacting the sample with a first type specific epitope ortype-cluster specific epitope under conditions which permitantigen-antibody binding; contacting the sample with a second typespecific epitope or type-cluster specific epitope under conditions whichpermit antigen-antibody binding; and determining whether antibodies inthe sample bind to either the first or second epitope.

Another aspect of the invention relates to polypeptides containing typespecific epitopes or type-cluster specific epitopes. The polypeptidesare derived from three different regions of the HCV genome. One set ofpolypeptides includes type specific epitope or type-cluster specificepitopes obtained from the HCV core region. This first set is foundbetween amino acid residues sixty-seven and eighty-four of HCV-1 andhomologous regions of other types of HCV. As used herein, the amino acidresidue abbreviations are as follows: A, alanine; I, isoleucine; L,leucine; M, methionine; F, phenylalanine; P, proline; W, tryptophan; V,valine; N, asparagine; C, cysteine; Q, glutamine;; G, glycine; S,serine; T, threonine; Y, tyrosine; R, arginine; H, histidine; K, lysine;D, aspartic acid; and E, glutamic acid.

The particular amino acid residue sequences derived from the core regionand subtypes from which they are derived are as follows:

1. PEGRTWAQ (SEQ ID NO:3), subtype 1a or 1b.

2. STGKSWGK (SEQ ID NO:4), subtype 2a or 2b.

3. SEGRSWAQ (SEQ ID NO:5), subtype 3a or 4.

Another set of polypeptides includes a type specific epitope obtainedfrom the HCV non-structural region 4 (NS4). This second set is foundbetween amino acid residues 1689-1718 of HCV-1 and homologous regions ofother types of HCV.

The particular amino acid residue sequences and types or subtypes fromwhich they are derived are as follows:

1. CSQHLPY (SEQ ID NO:6), subtype 1a.

2. CASHLPY (SEQ ID NO:7), subtype 1b.

3. CASRAAL (SEQ ID NO:8), subtype 2a or 2b.

4. CASKAAL (SEQ ID NO:23), subtype 2a or 2b.

Another set of polypeptides includes type specific epitope ortype-cluster specific epitopes obtained from the non-structural region 5(NS5) of a hepatitis C virus. This set is found between amino acidresidues 2281-2313 of HCV-1 and homologous regions of other types ofhepatitis C virus.

The particular amino acid residue sequences and types or subtypes fromwhich they are derived are as follows:

1. PDYEPPVVHG (SEQ ID NO:9), subtype 1a.

2. PDYVPPVVHG (SEQ ID NO:10), subtype 1b.

3. PDYQPATVAG (SEQ ID NO:11), subtype 2a.

4. PGYEPPTVLG (SEQ ID NO:12), subtype 2b.

5. FAQASPVW (SEQ ID NO:13), subtype 1a.

6. FPPQALPIW (SEQ ID NO:14), subtype 1b.

7. FPQALPAW (SEQ ID NO:15), subtype 2a.

8. FPPQALPPW (SEQ ID NO:16), subtype 2b.

Another aspect of the invention includes nucleic acid molecules encodingthe amino acid residue sequences of the type specific and type-clusterepitopes described. These nucleic acid molecules are useful as probesfor instance in Southern blots or other DNA recognition assays such asthe capture assay described in U.S. Pat. Nos. 4,868,105; and 5,124,246.

Another aspect of the invention includes nucleic acid moleculescomplementary to the nucleic acid sequences flanking regions encodingthe type specific and type-cluster specific epitopes. Such nucleic acidmolecules are useful in performing PCR to determine the genotype of aparticular HCV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the serotyping experimental strategy.

FIG. 2 is a flow diagram of the comprehensive epitope mapping strategy.

FIG. 3 (SEQ ID NO:1) is a compilation of graphs depicting the results ofepitope mapping of HCV 1a (Rodney).

FIG. 4 (SEQ ID NO:2) is a compilation of graphs depicting the results ofepitope mapping of HCV 2b (Nomoto).

DEFINITIONS

"Hepatitis C virus" or "HCV" refers to the viral species of whichpathogenic types cause NANBH, and attenuated types or defectiveinterfering particles derived therefrom. See generally, publicationscited in the section entitled "Background." The HCV genome is comprisedof RNA. RNA containing viruses have relatively high rates of spontaneousmutation reportedly on the order of 10⁻³ to 10⁻⁴ per incorporatednucleotide. Fields & Knipe (1986) "Fundamental Virology" (Raven Press,NY). Since heterogeneity and fluidity of genotype are inherent in RNAviruses, there are multiple types/subtypes, within the HCV species whichmay be virulent or avirulent. The propagation, identification,detection, and isolation of various HCV types or isolates is documentedin the literature. As depicted herein, all nucleotide and amino acidresidue sequences are from the HCV types noted. The number of the HCV-1genome and amino acid residue sequences is as descibed in Choo et al.(1990) Brit. Med. Bull., 46:423-441. The disclosure herein allows thediagnosis of the various types.

As used herein, "type" refers to HCVs that differ genotypically by morethan about 30%; "subtype" refers to HCVs that differ genotypically byabout 10-20% and "isolate" refers to HCVs that differ genotypically byabout less than 10%. "Typing" refers to distinguishing one type of HCVfrom another type.

Information on several different HCV types/subtypes is disclosed inInternational Publication No. WO 93/00365 particularly type or subtypeCDC/HCV1 (also called HCV-1). Information from one type or subtype, suchas a partial genomic or amino acid sequence, is sufficient to allowthose skilled in the art using standard techniques to isolate new typesof HCV. For example, several different types of HCV were screened asdescribed below. These types, which were obtained from a number of humansera (and from different geographical areas), were typed utilizing themethod and reagents described herein.

The genomic structure and the nucleotide sequence of HCV-1 genomic RNAhas been deduced. The genome appears to be single-stranded RNAcontaining 10,000 nucleotides. The genome is positive-stranded, andpossesses a continuous, translational open reading frame (ORF) thatencodes a polyprotein of about 3,000 amino acids. In the ORF, thestructural protein(s) appear to be encoded in approximately the firstquarter of the amino-terminus region, with the majority of thepolyprotein responsible for non-structural (NS) proteins. When comparedwith all known viral sequences, small but significant co-linearhomologies are observed with the non-structural (NS) proteins of theflavivirus family, and with the pestiviruses (which are now alsoconsidered to be part of the Flavivirus family).

Based upon the putative amino acid residues encoded in the nucleotidesequence of HCV-1 and other evidence, possible protein domains of theencoded HCV polyprotein, as well as the approximate boundaries, arepresented in Table 1.

                  TABLE 1                                                         ______________________________________                                                         Approximate Boundary                                         Putative Domain  (amino acid nos.)                                            ______________________________________                                        C(nucleocapsid protein)                                                                         1-191                                                       E.sub.1 (virion envelope protein)                                                              192-383                                                      E.sub.2 /NS1 (envelope?)                                                                       384-800                                                      NS2 (unknown function)                                                                          800-1050                                                    NS3 (protease?)  1050-1650                                                    NS4 (unknown function)                                                                         1651-2100                                                    NS5 (polymerase) 2100-3011 (end)                                              ______________________________________                                    

These domains are tentative. For example, the E2/NS1-NS2 border isprobably in the 750-810 region, and NS3-NS4 border is about 1640-1650.There is also evidence that the 191 amino acid (aa) version of C is aprecursor that is further processed to about 170 aa in length, and thatthe NS2, NS4 and NS5 proteins are each further processed into two matureproteins.

Different types of HCV are defined according to various criteria suchas, for example, an ORF of approximately 9,000 nucleotides toapproximately 12,000 nucleotides, encoding a polyprotein similar in sizeto that of HCV-1, an encoded polyprotein of similar hydrophobic and/orantigenic character to that of HCV-1, and the presence of co-linearpolypeptide sequences that are conserved with HCV-1.

The following parameters of nucleic acid homology and amino acidhomology are applicable, either alone or in combination, in identifyingHCV types. Generally, as described above, different types of HCV areabout 70% homologous whereas subtypes are about 80-90% homologous andisolates are about 90% homologous.

As used herein, a polynucleotide "derived from" a designated sequencerefers to a polynucleotide sequence which is comprised of a sequence ofat least about 6 nucleotides, preferably at least about 8 nucleotides,more preferably at least about 10-12 nucleotides, and even morepreferably at least about 15-20 nucleotides corresponding to a region ofthe designated nucleotide sequence. "Corresponding" means homologous toor complementary to the designated sequence. Preferably, the sequence ofthe region from which the polynucleotide is derived is homologous to orcomplementary to a sequence which is unique to an HCV genome.Hybridization techniques for determining the complementarity of nucleicacid sequences are known in the art. See, for example, Maniatis et al.(1982). In addition, mismatches of duplex polynucleotides formed byhybridization can be determined by known techniques, including forexample, digestion with a nuclease such as S1 that specifically digestssingle-stranded areas in duplex polynucleotides. Regions from whichtypical DNA sequences may be "derived" include but are not limited to,for example, regions encoding type specific epitopes, as well asnon-transcribed and/or non-translated regions.

The derived polynucleotide is not necessarily physically derived fromthe nucleotide sequence shown, but may be generated in any manner,including for example, chemical synthesis or DNA replication or reversetranscription or transcription. In addition, combinations of regionscorresponding to that of the designated sequence may be modified in waysknown in the art to be consistent with an intended use.

Similarly, a polypeptide or amino acid sequence "derived from" adesignated amino acid or nucleic acid sequence refers to a polypeptidehaving an amino acid sequence identical to that of a polypeptide encodedin the sequence, or a portion thereof wherein the portion consists of atleast 3-5 amino acids, and more preferably at least 8-10 amino acids,and even more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.This terminology also includes a polypeptide expressed from a designatednucleic acid sequence.

A recombinant or derived polypeptide is not necessarily translated froma designated nucleic acid sequence; it may be generated in any manner,including for example, chemical synthesis, or expression of arecombinant expression system, or isolation from HCV, including mutatedHCV. The polypeptides described herein are generally relatively shortand are thus most easily chemically synthesized.

A recombinant or derived polypeptide may include one or more analogs ofamino acids or unnatural amino acids in its sequence. Methods ofinserting analogs of amino acids into a sequence are known in the art.It also may include one or more labels, which are known to those ofskill in the art. A detailed description of analogs and "Mimotopes™" isfound in commonly owned, co-pending U.S. patent application Ser. No.07/972,755.

Peptide analogs include deletions, additions, substitutions ormodifications thereof which retain the HCV typing capability. Preferred"substitutions" are those which are conservative, i.e., wherein aresidue is replaced by another of the same general type. As is wellunderstood, naturally-occurring amino acids can be subclassified asacidic, basic, neutral and polar, or neutral and nonpolar. Furthermore,three of the encoded amino acids are aromatic. It is generally preferredthat encoded polypeptides differing from the natural epitope containsubstituted codons for amino acids which are from the same group as thatof the amino acid replaced. Thus, in general, the basic amino acids Lys,Arg, and His are interchangeable; the acidic amino acids aspartic andglutamic are interchangeable; the neutral polar amino acids Ser, Thr,Cys, Gln, and Asn are interchangeable; the nonpolar aliphatic aminoacids Gly, Ala, Val, Ile, and Leu are conservative with respect to eachother (but because of size, Gly and Ala are more closely related andVal, Ile and Leu are more closely related), and the aromatic amino acidsPhe, Trp, and Tyr are interchangeable. While proline is a nonpolarneutral amino acid, it represents difficulties because of its effects onconformation, and substitutions by or for proline are not preferred,except when the same or similar conformational results can be obtained.Polar amino acids which represent conservative changes include Ser, Thr,Gln, Asn; and to a lesser extent, Met. In addition, although classifiedin different categories, Ala, Gly, and Ser seem to be interchangeable,and Cys additionally fits into this group, or may be classified with thepolar neutral amino acids.

It should further be noted that if the polypeptides are madesynthetically, substitutions by amino acids which are not encoded by thegene may also be made. Alternative residues include, for example, theomega amino acids of the formula H₂ N(CH₂)_(n) COOH wherein n is 2-6.These are neutral, nonpolar amino acids, as are sarcosine (Sar), t-butylalanine (t-BuA), t-butyl glycine (t-BuG), N-methyl Ile (N-MeIle), andnorleucine (Nle). Phenyl glycine, for example, can be substituted forTrp, Tyr or Phe an aromatic neutral amino acid; citrulline (Cit) andmethionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine(Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, andornithine (Orn) is basic. The conformation conferring properties of theproline residues may be retained if one or more of these is substitutedby hydroxyproline (Hyp).

The term "recombinant polynucleotide" as used herein intends apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature.

The term "polynucleotide" as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNAand RNA. It also incudes known types of modifications, for example,labels which are known in the art, methylation, "caps," substitution ofone or more naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins including but not limited to nucleases, toxins,antibodies, signal peptides and poly-L-lysine; those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylators, those with modified linkages (e.g., alphaanomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide. The polynucleotides described herein are relativelyshort and are thus most easily chemically synthesized.

A "purified" polypeptide refers to the polypeptide being in a state thatis substantially free of other polypeptides, i.e., in a composition thatcontains a minimum of about 50% by weight (desired polypeptide/totalpolypeptide in composition), preferably a minimum of about 70%, and evenmore preferably a minimum of about 90% of the desired polypeptide,without regard to nonproteinaceous materials in the composition.Techniques for purifying viral polypeptides are known in the art.Purified antibodies are similarly defined in the art.

As used herein, "epitope" refers to an antigenic determinant of apolypeptide. An epitope could comprise 3 or more amino acids that definethe binding site of an antibody. Generally an epitope consists of atleast 5 amino acids, and sometimes consists of at least 8 amino acids.Methods of epitope mapping are known in the art.

As used herein, "type specific epitope" refers to an epitope that isfound on one HCV type. A "type-cluster specific epitope" is found onmore than one but fewer than all HCV types. For instance, a particularepitope may be recognized by antibodies from a patient infected with HCV1 but not recognized by or recognized less efficiently by antibodiesfrom a patient infected with HCV 2. Similarly, a type-cluster specificepitope derived from HCV-3 may be recognized by antibodies from apatient infected with HCV-3 or HCV-4 but not by antibodies from apatient infected with HCV-1 or HCV-2. "Conserved epitopes" are thosewhich are recognized by antibodies specific to all HCV types.

A polypeptide is "immunologically reactive" with an antibody which bindsto the peptide due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly by the kinetics ofantibody binding, and/or by competition in binding using as competitorsknown polypeptides containing an epitope against which the antibody isdirected. The techniques for determining whether a polypeptide isimmunologically reactive with an antibody are known in the art.

As used herein, the term "antibody" refers to a polypeptide or group ofpolypeptides which are comprised of at least one antibody combiningsite. An "antibody combining site" or "binding domain" is formed fromthe folding of variable domains of an antibody molecule(s) to formthree-dimensional binding spaces with an internal surface shape andcharge distribution complementary to the features of an epitope of anantigen, which allows an immunological reaction with the antigen. Anantibody combining site may be formed from a heavy and/or a light chaindomain (V_(H) and V_(L), respectively), which form hypervariable loopswhich contribute to antigen binding. The term "antibody" includes, forexample, vertebrate antibodies, hybrid antibodies, chimeric antibodies,altered antibodies, univalent antibodies, the Fab proteins, and singledomain antibodies.

Antibodies specific to polypeptides can be made by any method known inthe art. For instance, the polypeptides are generally suspended in aphysiologically acceptable buffer, mixed with a suitable adjuvant andinjected into an animal. Methods of making polyclonal and monoclonalantibodies are known in the art and will not be described in detailherein.

The term "polypeptide" refers to a polymer of amino acids and does notrefer to a specific length of the product; thus, polypeptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post-expressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation, and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

"Treatment," as used herein, refers to prophylaxis and/or therapy.

An "individual," as used herein, refers to vertebrates, particularlymembers of the mammalian species, and includes, but is not limited to,animals (e.g., dogs, cats, cattle, swine, sheep, goat, rabbits, mice,rats, guinea pigs, etc.), and primates, including monkeys, chimps,baboons and humans.

As used herein, the "sense strand" of a nucleic acid contains thesequence that has sequence homology to that of mRNA. The "anti-sensestrand" contains a sequence which is complementary to that of the "sensestrand".

As used herein, a "positive stranded genome" of a virus is one in whichthe genome, whether RNA or DNA, is single-stranded and which encodes aviral polypeptide(s). Examples of positive stranded RNA viruses includeTogaviridae, Coronaviridae, Retroviridae, Picornaviridae, andCaliciviridae. Included also, are the Flaviviridae, which were formerlyclassified as Togaviradae. See Fields & Knipe (1986).

As used herein, "antibody containing body sample" refers to a componentof an individual's body which is a source of the antibodies of interest.Antibody containing body components are known in the art, and includebut are not limited to, for example, plasma, serum, spinal fluid, lymphfluid, the external sections of the respiratory, intestinal, andgenitourinary tracts, tears, saliva, milk, white blood cells, andmyelomas.

As used herein, a "biological sample" refers to a sample of tissue orfluid isolated from an individual, including, but not limited to, forexample, plasma, serum, spinal fluid, lymph fluid, the external sectionsof the skin, respiratory, intestinal and genitourinary tracts, tears,saliva, milk, blood cells, tumors, organs. Also included are samples ofin vitro cell culture constituents (including, but not limited to,conditioned medium resulting from the growth of cells in culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, polypeptide and nucleic acid synthesis, and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fitsch & Sambrook,"Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning, Volumes Iand II" (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gaited., 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higginseds. 1984); "Transcription and Translation" (B. D. Hames & S. J. Higginseds. 1984); "Animal Cell Culture" (R. I. Freshney ed. 1986);"Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "APractical Guide To Molecular Cloning" (1984); the series, "Methods inEnzymology" (Academic Press, Inc.); "Gene Transfer Vectors For MammalianCells" (J. H. Miller and M. P. Calos eds., 1987, Cold Spring HarborLaboratory), Meth. Enzymol., Vol. 154 and Vol. 155 (Wu and Grossman, andWu, eds., respectively), Mayer and Walker, eds. (1987), "ImmunochemicalMethods In Cell And Molecular Biology" (Academic Press, London); Scopes,(1987) "Protein Purification: Principles and Practice", Second Edition(Springer-Verlag, N.Y.); and "Handbook of Experimental Immunology",Volumes I-IV (D. M. Weir and C. C. Blackwell eds. 1986).

All patents, patent applications and publications mentioned herein, bothsupra and infra, are hereby incorporated herein by reference.

The invention includes methods for detecting HCV and identifyinginfection by different types of HCV. The invention also includespolypeptides and nucleic acid molecules for use in the methods.

The methods for detecting and typing infection by HCV include bothimmunoassays and nucleic acid identification by methods including butnot limited to, Southern blot analysis and polymerase chain reaction. Inorder to identify infection by HCV, a biological sample is incubatedwith one of the polypeptides described herein under conditions whichpermit antigen-antibody binding and a determination is made as towhether antibodies in the sample bind to the epitope found on thepolypeptide.

Immunoassay and Diagnostic Kits

The polypeptides containing the type specific epitopes and type-clusterspecific epitopes are useful in immunoassays to detect the presence ofHCV antibodies, or the presence of the virus and/or viral antigens, inbiological samples. Design of the immunoassays is subject to a greatdeal of variation, and many formats are known in the art. Theimmunoassay will utilize at least one type specific epitope ortype-cluster specific epitope. In one embodiment, the immunoassay uses acombination of type specific epitopes and/or type-cluster specificepitopes.

The polypeptides are useful for typing HCV by using the epitopes todetermine the presence of type specific or type-cluster specificantibodies. The polypeptides are also suitable for use in generatingtype specific or type-cluster specific antibodies that can then be usedin an immunoassay to distinguish between various types of HCV.

The polypeptides are derived from three different regions of the HCVgenome. One set of polypeptides includes a type or type cluster specificepitope obtained from the HCV core region. Another set of polypeptidesincludes a type or type cluster specific epitope obtained from the HCVnon-structural region 4 (NS4). Another set of polypeptides includes atype or type-cluster specific epitope obtained from the non-structuralregion 5 (NS5) of a hepatitis C virus. This set is found between aminoacid residues 2281-2313 of HCV-1 and homologous regions of other typesof hepatitis C virus.

The polypeptides are suitable for use in immunoassays for one or moreHCV types. In order to assay for one type the sample is contacted withone or more polypeptides containing a type-cluster specific epitopeunder conditions which permit antigen-antibody binding and determiningwhether antibodies in the sample bind to the epitope.

In an immunoassay to distinguish a particular type of HCV, a biologicalsample is obtained from an individual, contacted with a first typespecific epitope or type-cluster specific epitope under conditions whichpermit antigen-antibody binding; contacted with a second type specificepitope or type-cluster specific epitope under conditions which permitantigen-antibody binding and determining whether antibodies in thesample bind to either the first or second epitope. These steps can berepeated with any number of polypeptides containing type and/ortype-cluster specific epitopes.

Typically, an immunoassay for anti-HCV antibody(s) involves selectingand preparing the test sample suspected of containing the antibodies,such as a biological sample, then incubating it with the type specificepitope or type-cluster specific epitope under conditions that allowantigen-antibody complexes to form, and then detecting the formation ofsuch complexes. Suitable incubation conditions are well known in theart. The immunoassay may be, without limitations, in a heterogeneous orin a homogeneous format, and of a standard or competitive type.

In a heterogeneous format, the type specific epitope or type-clusterspecific epitope is typically bound to a solid support to facilitateseparation of the sample from the polypeptide after incubation. Examplesof solid supports that can be used include, but are not limited to,nitrocellulose (e.g., in membrane or microtiter well form), polyvinylchloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g.,in beads or microtiter plates), polyvinylidine fluoride (known asImmulon®), diazotized paper, nylon membranes, activated beads, andProtein A beads. For example, Dynatech Immulon® 1 or Immulon® 2microtiter plates or 0.25 inch polystyrene beads (Precision PlasticBall) can be used in the heterogeneous format. The solid supportcontaining the type specific epitope or type-cluster epitope istypically washed after separating it from the test sample, and prior todetection of bound antibodies. Both standard and competitive formats areknown in the art.

In a homogeneous format, the test sample is incubated with the typespecific epitope or type cluster epitope in solution. For example, itmay be under conditions that will precipitate any antigen-antibodycomplexes which are formed. Both standard and competitive formats forthese assays are known in the art.

In a standard format, the amount of HCV antibodies forming theantibody-type or -type-cluster specific epitope complex is directlymonitored. This may be accomplished by determining whether labeledanti-xenogeneic (e.g., anti-human) antibodies which recognize an epitopeon anti-HCV antibodies will bind due to complex formation. In acompetitive format, the amount of HCV antibodies in the sample isdeduced by monitoring the competitive effect on the binding of a knownamount of labeled antibody (or other competing ligand) in the complex.

In an inhibition assay, the ability of antibodies to bind topolypeptides containing various different type specific epitopes ortype-cluster specific epitopes is determined. The antibodies are firstexposed to polypeptides containing epitope(s) from one type ortype-cluster of HCV and then to polypeptides containing epitope(s) fromanother type or type-cluster of HCV. The process may be repeated foradditional types of HCV.

Complexes formed comprising anti-HCV antibody (or, in the case ofcompetitive assays, the amount of competing antibody) are detected byany of a number of known techniques, depending on the format. Forexample, unlabeled HCV antibodies in the complex may be detected using aconjugate of antixenogeneic Ig complexed with a label, (e.g., an enzymelabel).

In typical immunoassays, the test sample, typically a biological sample,is incubated with polypeptides containing one or more type specificepitopes or type-cluster specific epitopes under conditions that allowthe formation of antigen-antibody complexes. Various formats can beemployed. For example, a "sandwich assay" may be employed, whereantibody bound to a solid support is incubated with the test sample;washed; incubated with a second, labeled antibody to the analyte, andthe support is washed again. Analyte is detected by determining if thesecond antibody is bound to the support. In a competitive format, whichcan be either heterogeneous or homogeneous, a test sample is usuallyincubated with antibody and a labeled, competing antigen is alsoincubated, either sequentially or simultaneously. These and otherformats are well known in the art.

Antibodies directed against the type specific epitopes or type-clusterspecific epitopes can be used in immunoassays for the detection of viralantigens in patients with HCV caused NANBH, and in infectious blooddonors. Moreover, these antibodies may be extremely useful in detectingacute-phase donors and patients.

An immunoassay may use, for example, a monoclonal antibody directedtowards a type specific epitope or type-cluster specific epitopes, acombination of monoclonal antibodies directed towards epitopes of oneviral antigen, monoclonal antibodies directed towards epitopes ofdifferent viral antigens, polyclonal antibodies directed towards thesame viral antigen, or polyclonal antibodies directed towards differentviral antigens. Protocols may be based, for example, upon competition,or direct reaction, or sandwich type assays. Protocols may also, forexample, use solid supports, or may be by immunoprecipitation. Mostassays involve the use of labeled antibody or polypeptide; the labelsmay be, but are not limited to, enzymatic, fluorescent,chemiluminescent, radioactive, or dye molecules. Assays which amplifythe signals from the probe are also known; examples of which are assayswhich utilize biotin and avidin, and enzyme-labeled and mediatedimmunoassays, such as ELISA assays.

The invention further includes nucleic acid molecules encoding the aminoacid residue sequences of the type specific epitopes and type-clusterspecific epitopes described. These nucleic acid molecules are useful asprobes for instance in Southern blots or other DNA recognition assayssuch as the capture assay described in U.S. Pat. Nos. 4,868,105 and5,124,246.

The studies on antigenic mapping by expression of HCV cDNAs showed thata number of clones containing these cDNAS expressed polypeptides whichwere immunologically reactive with serum from individuals exhibitingNANBH. No single polypeptide was immunologically reactive with all sera.Five of these polypeptides were very immunogenic in that antibodies tothe HCV epitopes in these polypeptides were detected in many differentpatient sera, although the overlap in detection was not complete. Thus,the results on the immunogenicity of the polypeptides encoded in thevarious clones suggest that efficient detection systems for HCVinfection may include the use of panels of epitopes. The epitopes in thepanel may be constructed into one or multiple polypeptides. The assaysfor the varying epitopes may be sequential or simultaneous.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the polypeptides of the invention containing type specificepitopes and type-cluster epitopes or antibodies directed against typespecific epitopes and type-cluster specific epitopes in suitablecontainers, along with the remaining reagents and materials required forthe conduct of the assay, as well as a suitable set of assayinstructions.

The invention further includes nucleic acid molecules complementary tothe nucleic acid sequences flanking regions encoding the type specificepitopes and type-cluster specific epitopes. Such nucleic acid moleculesare useful in performing PCR to determine the genotype of a particularHCV.

It should be noted that there are variable and hypervariable regionswithin the HCV genome; therefore, the homology in these regions isexpected to be significantly less than that in the overall genome.

The techniques for determining nucleic acid and amino acid sequencehomology are known in the art. For example, the amino acid sequence maybe determined directly and compared to the sequences provided herein.Alternatively the nucleotide sequence of the genomic material of theputative HCV may be determined (usually via a cDNA intermediate), theamino acid sequence encoded therein can be determined, and thecorresponding regions compared.

The foregoing discussion and examples only illustrate the invention,persons of ordinary skill in the art will appreciate that the inventioncan be implemented in other ways, and the invention is defined solely byreference to the claims.

EXAMPLE 1 Comparison of Major Epitopes of Various Different Types of HCV

The amino acid residue homology between different types and subtypes ofHCV was compared for various regions. The subtype of HCV is as describedby Simmonds phylogenetic analysis. The amino acid sequence numberingcorresponds to that described for the prototype HCV-1 sequence. Choo etal. (1989) Table 2 shows the percent amino acid residue homology for NS4region type specific epitopes and type-cluster specific epitopes and theconserved major epitope. Table 3 shows the amino acid residue homologybetween two type specific epitopes or type-cluster specific of the NS5region. Table 4 shows the percent amino acid residue homology for coreregion conserved major epitopes and type specific epitopes.

                  TABLE 2                                                         ______________________________________                                        Amino Acid Homologies (%) Between Different HCV Subtypes                                         NS4 region type                                                                            NS4 region                                                       specific major                                                                             conserved major                               HCV   Example types                                                                              epitopes     epitope                                       subtype                                                                             abbreviation (1689-1718 aa)*                                                                            (1910-1936 aa)*                               ______________________________________                                        1a    HCV-1 (1a) vs (1a)                                                                         100%         100%                                          1b    HCV-J (1a) vs (1b)                                                                         83%          100%                                          2a    HCV-J6 (1a) vs (2a)                                                                        47%           93%                                          2b    RCV-J8 (1a) vs (2b)                                                                        43%           93%                                          ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Amino Acid Homologies (%) Between Different HCV Subtypes                                          NS4 region type                                                                           NS5 region type                                                   specific major                                                                            specific major                                HCV   Example types epitopes    epitope                                       subtype                                                                             abbreviation  (2281-2313 aa)*                                                                           (2673-2707 aa)*                               ______________________________________                                        1a    HCV-1 (1a) vs (1a)                                                                          100%        100%                                          1b    HCV-J (1a) vs (lb)                                                                          76%         89%                                           2a    HCV-J6 (1a) vs (2a)                                                                         70%         83%                                           2b    HCV-J8 (1a) vs (2b)                                                                         73%         83%                                           3a    HCV-E-b1 (1a) vs (3a)     77%                                           3b    HCV-Tb (1a) vs (3b)       83%                                           ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Amino Acid Residue Homology (%) Between Different HCV Subtypes                                     Core region Core region type                                                  conserved major                                                                           specific major                               HCV   Example types  epitopes    epitope                                      subtype                                                                             abbreviation   (10-45 aa)* (67-84 aa)*                                  ______________________________________                                        1a    HCV-1 (1a) vs (1a)                                                                           100%        100%                                         1b    HCV-J (1a) vs (1b)                                                                           98%         100%                                         2a    HCV-J6 (1a) vs (2a)                                                                          98%         61%                                          2b    HCV-JB (1a) vs (2b)                                                                          98%         61%                                          3a    HCV-E-b1 (1a) vs (3a)                                                                        93%         89%                                          4     HCV-EG-21 (1a) vs (4)                                                                        98%         83%                                          ______________________________________                                    

EXAMPLE 2 Peptide Synthesis

Two sets of polypeptides were synthesized. The first set was designed toperform epitope mapping of HCV-1 and the second set was designed todetermine which epitopes identified in the epitope mapping studiescontained type specific epitopes. In the first set of polypeptides,sixty-four sets (in duplicate) of overlapping octapeptides weresynthesized by Mimotopes across the entire HCV-1 polyprotein (3011 aminoacid residues).

The second set of polypeptides were made according to the methoddescribed by Geysen (1990) J. Trop. Med. Pub. Health, 21:523-533; andMerrifield (1963) J. Am. Chem. Soc., 85:2149-2154.

In the second set of polypeptides, four antigenic regions whichrepresent the major epitopes of non-conservative sequences in HCV-1 fromcore, NS4, NS5 and their corresponding sequences from HCV subtypes 1b,2a, 3a and type 4 were selected for type specific epitope synthesis. Thesequence from core was selected from the less conserved region of aminoacid residues 67-84. The sequence from the NS4 region was selected fromthe amino acid residue region 1689-1718. The sequences selected from theNS5 region were from the amino acid residue regions 2281-2313 and2673-2707.

EXAMPLE 3 Biological Samples

In order to determine the effectiveness of the polypeptides indistinguishing between antibodies specific to different types of HCV,antisera were obtained from twenty-four chronic NANBH patients fromdifferent areas of the world including the United States east coast andwest coast, Japan, western European countries, southern Europeancountries and South Africa. The viral RNA isolation, cDNA synthesis, PCRamplification, DNA sequencing and oligonucleotide probe hybridizationwere performed as described by Cha et al. (1992) Proc. Natl. Acad. Sci.USA, 89:7144-7148.

EXAMPLE 4 Epitope Mapping Procedures

In order to determine which regions of HCV contain epitopes, whethergroup specific or conserved, the entire HCV-1 polyprotein was subject toepitope mapping. The method used is essentially as outlined in FIG. 2.

Sixty-four sets (in duplicate) of overlapping octapeptides weresynthesized by Mimotopes across the entire HCV-1 polyprotein (3011 aminoacids). A panel of 40 samples which contain 25 United States HCVantibody reactive samples, 9 Japan HCV reactive samples and 6 HCVnon-reactive negative control samples were selected for epitope mappingand cluster analysis. The immunoassays were performed using standardELISA procedures.

The criteria for identifying the major epitopes were based on theantibody reaction frequency and the antibody reaction intensity (titer)to these epitopes. The results are presented in FIGS. 3 and 4 (SEQ IDNO:1 and SEQ ID NO:2).

EXAMPLE 5 Polypeptide Derived Enzyme-linked Immunosorbent Assays

In order to determine the optimal immunoassay utilizing the polypeptidesdescribed in Example 2, two separate types of polypeptide derivedenzyme-linked immunosorbent assay (ELISA) were run and the results werecompared. The first type of ELISA was the Nunc MaxiSorb™ on which thepolypeptides are simply adsorbed and the second type utilized NuncCovalink NH™ on which the polypeptides are covalently linked. Formationof amide bonds between carboxylic acids and amines is initiated by theaddition of carbodiimide. To reduce hydrolysis, the active ester can bemade by adding N-hydroxy-succinimide (NHS) to the above conjugationprocedures.

The microtiter plates for the first type of ELISA was performed asfollows. The polypeptides were placed in the wells of the Nunc MaxiSorb™microtiter plates at a concentration of 1 μg/well in 100 μl of phosphatebuffered saline (PBS). The polypeptides were allowed to absorb overnightat room temperature. The microtiter plates were then washed four timeswith PBS without detergent. The wells were then post-coated with 220 μlSuperblock® (Pierce) for one hour and then aspirated without furtherwashing and vacuum dried.

The assay was performed as follows. The sera sample size of 5 μl wasadded to the wells with 100 μl of 5% nonfat milk and incubated for onehour at 37° C. The wells were then washed five times in PBS with 0.05%Tween. A conjugate of affinity purified goat anti-human IgG labeled withhorse radish peroxidase (Jackson Laboratories) was then used todetermine the degree of binding of human antibodies to the polypeptides.The conjugate was previously diluted to 5% IgG in 150 mM NaCl, PBS, 5%horse serum (heat denatured). 100 μl of the conjugate was placed in thewells and incubated for one hour at 37° C. The wells were then washedfive times with PBS/Tween and OPD [o-phenylene-diamine-2HCl, one tabletper developer buffer (citrate phosphate buffered 0.02% H₂ O₂); Sigma]for thirty minutes at room temperature and the Abs at 492 nm and 620 nmwere determined. The cut off was determined from 200 random (normal)samples where seven standard deviations from the mean or about 0.45.

The microtiter plates for the second type of ELISA was performed asfollows. The polypeptides were placed in the wells of the Nunc MaxiSorb™microtiter plates at a concentration of 10 mg/well in 50 μl of water. 25μl of 0.1 M NHS (sulfo-N-hydrosuccinimide, Pierce) and 25 μl of 0.1 MEDC [1-Ethyl-3(3-dimethylaminopropylcarbodiimide) Sigma] were added tothe polypeptides and mixed at room temperature for 30 minutes on arocking platform. The entire contents were then added to 52 ml of icecold 0.1 M Na Carbonate pH 8.6. 100 μl of the mixture was used to coatthe wells of the microtiter plates and then incubated at 4° C. for 30minutes. The plates were then washed four times with PBS/0.1% TritonX-100. The plates were then treated with Superblock® and the assay wasperformed as described above.

The results obtained are presented in the following Tables.

                                      TABLE 5                                     __________________________________________________________________________    Serotyping Epitope Analysis from Different HCV Types                          Sequence Region:                                                                            NS4 (1689-1718)                                                                                  Peptide                                      Sample ID      EIA                  Type dependent sequences                  __________________________________________________________________________     ##STR1##                                                                     __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Serotyping Epitope Analysis from Different HCV Types                          __________________________________________________________________________    Sequence Region:                                                                            N54 (1689-1718)                                                 Sample Description                                                                          Peptide EIA                                                                             Peptide from                                                                             Type dependent sequences                                 Signal/Cutoff                                                                           different HCV                                                                 types                                                 (I) Paid Donor Samples             SGKPAIIPDREVLYREFDEMEECSQHLPYI                                                                   (SEQ ID NO:17)                                  cp402-10(la)                                                                             SGKPAIIPDREVLYREFDEMEECSQHLPYI                                                                   (SEQ ID NO:17)                                  cp402-11(lb)                                                                             SGRPAVIPDREVLYQFDEMEECASHLPYI                                                                    (SEQ ID NO:18)                                  cp402-12(2a)                                                                             NQRAWAPDKEVLYEAFDEMEECAS                                                                         (SEQ ID NO:19)                                  cp402-13(2b)                                                                             NDRWVVVAPDKEILYEAFDEMEECASKAALI                                                                  (SEQ ID NO:20)                                              * *         *          *                  Paid Donor Samples                                                            FF25910 (la)   5.54     cp402-10(la)                                                         9.71     cp402-11(lb)                                                         8.28     cp402-12(2a)                                                         6.63     cp402-13(2b)                                                         6.87     sodCl00 ELISA(la)                                     React with common epitopes la, lb, 2a & 2b                                                                               PDREVLY    (SEQ ID NO:21)                                                       K I                              (II) Sample react with type-specific epitopes                                 FF25931        5.74     cp402-10(la)                                                         7.43     cp402-11(lb)                                                         8.89     cp402-12(2a)                                                        14.5      cp402-13(2b)                                                         7.25     sodCl00 ELISA(la)                                     React with common epitope for la, lb, 2a & 2b                                                                            PDREVLY    (SEQ ID NO:22)                                                       K I                              __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Serotyping Epitope Analysis from Different HCV Types                          Sequence Region:                                                                       NS4 (1689-1718)                                                                         Peptides from                                              Sample   Peptide EIA                                                                             different HCV                                              Description                                                                            Signal/Cutoff                                                                           types      Type dependent sequences                        __________________________________________________________________________     ##STR2##                                                                     __________________________________________________________________________

                                      TABLE 8                                     __________________________________________________________________________    Serotyping Epitope Analysis from Different HCV Types                          Sequence Region:                                                                         NS5 (2673-2707)                                                                          Peptides from                                           Sample                different HCV                                           Description                                                                              Peptide EIA S/CO                                                                         types       Type dependent sequences                    __________________________________________________________________________     ##STR3##                                                                     __________________________________________________________________________

                                      TABLE 9                                     __________________________________________________________________________    Serotyping Epitope Analysis from Different HCV Types                          Sequence Region:                                                                         NS5 (2673-2707)                                                                          Peptides from                                           Sample                different HCV                                           Description                                                                              Peptide EIA S/CO                                                                         types          Type dependent sequences                 __________________________________________________________________________     ##STR4##                                                                     __________________________________________________________________________

                                      TABLE 10                                    __________________________________________________________________________    Summary of HCV Major type Specific Epitopes in Core, N54 and NS5              __________________________________________________________________________    Regions                                                                       Core Region:                                                                  Major Conserved Epitopes                                                                      type Specific Epitopes                                                                              Conserved Epitopes                      (15-45)         (67-84)               (67-84)                                 TNRRPQDVKFPGGGQIVGGVY                                                                         HCV-la & 1b-PEGRTWAQ(SEQ ID NO:31)                                                                  PGYPWP(SEQ ID NO:32)(1a, lb, 2a,                                              2b, 3a)                                 LLPRRGPRLG(HCV-1)                                                                             HCV-2a & 2b-STGKSWGK(SEQ ID NO:33)                            (SEQ ID NO:30)  HCV-3A or 4-SEGRSWAQ(SEQ ID NO:34)                                                                     F(4)                                 NS4 Region:                                                                   Major Conserved Epitopes                                                                      type Specific Epitopes                                                                              Conserved Epitopes                      (1925-1935)     (1689-1718)           (1689-1718)                             RGNHVSPTHYV(HCV-1)                                                                            HCV-1a-------CSQHLPYA(SEQ ID NO:36)                                                                 PDREVL(SEQ ID NO:37)(1a, 1b)            (SEQ ID NO:35)  HCV-lb-------CASHLPY(SEQ ID NO:38)                                                                    K I(2a, 2b)                                           HCV-2a & 2b-CASRAAL(SEQ ID NO:39)                                                            K                                              NS5 Region:                                                                   Conserved Epitopes                                                                            type Specific Epitopes                                        (2288-2294)     (2281-2313)                                                   WARPDYN         HCV-1a & 1b-PDYEPPVVHG(SEQ ID NO:41)                          (SEQ ID NO:40                  V                                                              HCV-2a--------PDYQPATVAG(SEQ ID NO:42)                                        HCV-2b--------PGYEPPTVLG(SEQ ID NO:43)                                        HCV-1a--------FAQALPVW(SEQ ID NO:44)                                          HCV-1b,2a&2b-FPPQALPIW(SEQ ID NO:45)                                                              A                                                                             P                                         Conserved Epitopes                                                            (2673-2707)                                                                   RGENCGYRRCRASGVLTT-HCV-1a(SEQ ID NO:46)                                                               KGAQCGYRRCRASGVLPT-HCV-3a(SEQ ID NO:49)               K QT  SEQ ID NO:47)HCV-1b, 2a                                                                                         *                                     K QS (SEQ ID NO:48)HCV-3b                                                                             KGQSCGYRRCRASGVFTT-HCV-2b(SEQ ID NO:50)                                                      *                                      __________________________________________________________________________

                                      TABLE 11                                    __________________________________________________________________________    Serotyping Epitope Analysis from Different Types of HCV Sequences (I)         Chronic NANBH from Paid                                                       Donors Sequence Region: Core (67-84)                                          Sample ID                                                                            Peptide EIA S/CO                                                                         Peptide (HCV type)                                                                          Type Dependent Sequences                      __________________________________________________________________________     ##STR5##                                                                     __________________________________________________________________________

                                      TABLE 12                                    __________________________________________________________________________    Serotyping Epitope Analysis from Different Types of HCV Sequences (I)         Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)               Sample ID Peptide EIA S/CO Peptide                                            (HCV Type)                      Type Dependent Sequences                      __________________________________________________________________________     ##STR6##                                                                     __________________________________________________________________________

                                      TABLE 13                                    __________________________________________________________________________    Serotyping Epitope Analysis from Different Types of HCV Sequences (I)         Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)               Sample ID Peptide EIA S/CO Peptide                                            (HCV Type)              Type Dependent Sequences                              __________________________________________________________________________     ##STR7##                                                                     __________________________________________________________________________

                                      TABLE 14                                    __________________________________________________________________________    Serotyping Epitope Analysis from Different Types of HCV Sequences (I)         Chronic NANBH from Paid Donors Sequence Region: NS5 (2281-2313)               Sample ID Peptide EIA S/CO Peptide                                            (HCV Type)                Type Dependent Sequences                            __________________________________________________________________________     ##STR8##                                                                     __________________________________________________________________________

EXAMPLE 6 Inhibition Assay

The inhibition assays were performed to determine whether the peptidescould compete with each other for binding to antibodies. Three sets ofshort polypeptides from the core, NS4 and NS5 regions of different typesof HCV sequences were synthesized. These polypeptides cover the sequenceregions from amino acid 1689-1695, 1696-1702 and 1711-1717. Theinhibition assays were performed by addition of 10 μg of the abovepolypeptides to the sample and incubation at 37° C. for one hour andthen performance of the ELISA assays as described above. If inhibitionwas found to be more than 50% of antibody binding, the polypeptide wasconsidered to be inhibitory. The results obtained are presented in thefollowing Table.

                                      TABLE 15                                    __________________________________________________________________________    Inhibition Analysis                                                           Polypeptide          Region Amino Acids                                                                          Virus type Inhibition                      __________________________________________________________________________    SQHLPY(SEQ ID NO:66) NS4 Region01                                                                         1712-1717                                                                            1a                                         ASRAAL(SEQ ID NO:67)    CHIEN-102                                                                         1712-1717                                                                            2a                                         ASKAAL(SEQ ID NO:68)    CHIEN-103                                                                         1712-1717                                                                            2b                                         PDREVLY(SEQ ID NO:69)   CHIEN-104                                                                         1696-1972                                                                            1a, 1b, 2a                                 PDYRPPVVHG(SEQ ID NO:70)CHIEN-105                                                                  NS5 Region                                                                           2304-2313                                                                            1a                                         PDYQPATVAG(SEQ ID NO:71)CHIEN-106                                                                         2304-2313                                                                            2a                                         FAQALPVW(SEQ ID NO:72)  CHIEN-107                                                                         2281-2288                                                                            1a                                         FPPQALPPW(SEQ ID NO:73) CHIEN-108                                                                         2281-2288                                                                            2b                                         STGKSWGK(SEQ ID NO:74)  CHIEN-108                                                                  Core   71-78  2a & 2b                                    SEGRSWAQ(SEQ ID NO:75)  CHIEN-110                                                                  Core   71-78  4                                          __________________________________________________________________________

    TABLE 16       - HCV Major Type Specific Epitopes in Core, NS4 & NS5 Regions       Major Conserved Type Specific Conserved       Epitopes Epitopes Epitopes      ##STR9##

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 75                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Val Cys Ala Arg Ala Gln Ala Pro Pro Pro Se - #r Trp Asp Gln Met        Trp                                                                             1               5   - #                10  - #                15              - - Lys Cys Leu Ile Arg Leu Lys Pro Thr Leu Hi - #s Gly Pro Thr Pro Leu                  20      - #            25      - #            30                   - - Leu Tyr Arg Leu Gly Ala Val Gln Asn Glu Il - #e Thr Leu Thr His Pro              35          - #        40          - #        45                       - - Val Thr Lys Tyr Ile Met Thr Cys Met Ser Al - #a Asp Leu Glu Val Val          50              - #    55              - #    60                           - - Thr Ser Thr Trp Val Leu Val Gly Gly Val Le - #u Ala Ala Leu Ala Ala      65                  - #70                  - #75                  - #80        - - Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Va - #l Gly Arg Val Val Leu                      85  - #                90  - #                95               - - Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Gl - #u Val Leu Tyr Arg Glu                  100      - #           105      - #           110                  - - Phe Asp Glu Met Glu Glu Cys Ser Gln His Le - #u Pro Tyr Ile Glu Gln              115          - #       120          - #       125                      - - Gly Met Met Leu Ala Glu Gln Phe Lys Gln Ly - #s Ala Leu Gly Leu Leu          130              - #   135              - #   140                          - - Gln Thr Ala Ser Arg Gln                                                  145                 1 - #50                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Val Cys Ala Arg Ala Lys Ala Pro Pro Pro Se - #r Trp Asp Val Met Trp      1               5   - #                10  - #                15               - - Lys Cys Leu Ala Arg Leu Lys Pro Thr Leu Il - #e Gly Pro Thr Pro Leu                  20      - #            25      - #            30                   - - Leu Tyr Arg Leu Gly Ala Val Thr Asn Glu Va - #l Thr Leu Thr His Pro              35          - #        40          - #        45                       - - Val Thr Lys Thr Ile Ala Thr Cys Met Ser Va - #l Asn Leu Glu Ile Met          50              - #    55              - #    60                           - - Thr Ser Thr Trp Val Leu Ala Gly Gly Val Le - #u Ala Ala Val Ala Ala      65                  - #70                  - #75                  - #80        - - Tyr Cys Leu Ala Thr Gly Cys Val Ser Ile Il - #e Gly Arg Leu His Leu                      85  - #                90  - #                95               - - Asn Asp Gln Val Val Val Thr Pro Asp Lys Gl - #u Ile Leu Tyr Glu Ala                  100      - #           105      - #           110                  - - Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Al - #a Ala Leu Ile Glu Glu              115          - #       120          - #       125                      - - Gly Gln Arg Met Ala Glu Met Leu Lys Ser Ly - #s Ile Gln Gly Leu Leu          130              - #   135              - #   140                          - - Gln Gln Ala Thr Arg Gln Ala Gln Asp Ile Gl - #n                          145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Pro Glu Gly Arg Thr Trp Ala Gln                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ser Thr Gly Lys Ser Trp Gly Lys                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Ser Glu Gly Arg Ser Trp Ala Gln                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Cys Ser Gln His Leu Pro Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Cys Ala Ser His Leu Pro Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Cys Ala Ser Arg Ala Ala Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Pro Asp Tyr Glu Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Pro Asp Tyr Val Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Pro Gly Tyr Glu Pro Pro Thr Val Leu Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Phe Ala Gln Ala Ser Pro Val Trp                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Phe Pro Pro Gln Ala Leu Pro Ile Trp                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Phe Pro Pro Gln Ala Leu Pro Ala Trp                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Phe Pro Pro Gln Ala Leu Pro Pro Trp                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Gl - #u Val Leu Tyr Arg Glu      1               5   - #                10  - #                15               - - Phe Asp Glu Met Glu Glu Cys Ser Gln His Le - #u Pro Tyr Ile                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Ser Gly Arg Pro Ala Val Ile Pro Asp Arg Gl - #u Val Leu Tyr Gln Phe      1               5   - #                10  - #                15               - - Asp Glu Met Glu Glu Cys Ala Ser His Leu Pr - #o Tyr Ile                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Asn Gln Arg Ala Val Val Ala Pro Asp Lys Gl - #u Val Leu Tyr Glu Ala      1               5   - #                10  - #                15               - - Phe Asp Glu Met Glu Glu Cys Ala Ser Arg Al - #a Ala Leu Ile                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Asn Asp Arg Val Val Val Ala Pro Asp Lys Gl - #u Ile Leu Tyr Glu Ala      1               5   - #                10  - #                15               - - Phe Asp Glu Met Glu Glu Cys Ala Ser Lys Al - #a Ala Leu Ile                          20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "This position is either R                        or K."                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "This position is either V                        or I."                                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Pro Asp Xaa Glu Xaa Leu Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Cys Ala Ser Arg Ala Ala Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Cys Ala Ser Lys Ala Ala Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Leu Thr Asn      1               5   - #                10  - #                15               - - Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Leu Thr Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Met Phe Asn      1               5   - #                10  - #                15               - - Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Leu Thr Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Met Phe Asn      1               5   - #                10  - #                15               - - Ser Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Leu Thr Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Ile Ser Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Met Phe Asn      1               5   - #                10  - #                15               - - Ser Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Leu Thr Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Ile His Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Met Thr Asn      1               5   - #                10  - #                15               - - Ser Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Phe Thr Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Ile Ser Ser Leu Thr Glu Arg Leu Tyr Cys Gl - #y Gly Pro Met Phe Asn      1               5   - #                10  - #                15               - - Ser Lys Gly Ala Gln Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val                  20      - #            25      - #            30                   - - Leu Pro Thr                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pr - #o Gly Gly Gly Gln Ile      1               5   - #                10  - #                15               - - Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gl - #y Pro Arg Leu Gly                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Pro Glu Gly Arg Thr Trp Ala Gln                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Pro Gly Tyr Pro Trp Pro                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Ser Thr Gly Lys Ser Trp Gly Lys                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Ser Glu Gly Arg Ser Trp Ala Gln                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Arg Gly Asn His Val Ser Pro Thr His Tyr Va - #l                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Cys Ser Gln His Leu Pro Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/note= "This position is R or K."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/note= "This position is V or I."          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Pro Asp Xaa Glu Xaa Leu Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Cys Ala Ser His Leu Pro Tyr                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "This position is R or K."          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Cys Ala Ser Xaa Ala Ala Leu                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Trp Ala Arg Pro Asp Tyr Asn                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/note= "This position is E or V."          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Pro Asp Tyr Xaa Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - Pro Gly Tyr Glu Pro Pro Thr Val Leu Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Phe Ala Gln Ala Leu Pro Val Trp                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/note= "This position is I or A or                       P."                                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Phe Pro Pro Gln Ala Leu Pro Xaa Trp                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Ar - #g Ala Ser Gly Val Leu      1               5   - #                10  - #                15               - - Thr Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - Lys Gly Gln Thr Cys Gly Tyr Arg Arg Cys Ar - #g Ala Ser Gly Val Leu      1               5   - #                10  - #                15               - - Thr Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cys Ar - #g Ala Ser Gly Val Leu      1               5   - #                10  - #                15               - - Thr Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - Lys Gly Ala Gln Cys Gly Tyr Arg Arg Cys Ar - #g Ala Ser Gly Val Leu      1               5   - #                10  - #                15               - - Pro Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - Lys Gly Gln Ser Cys Gly Tyr Arg Arg Cys Ar - #g Ala Ser Gly Val Phe      1               5   - #                10  - #                15               - - Thr Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Al - #a Gln Pro Gly Tyr Pro      1               5   - #                10  - #                15               - - Trp Pro                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gl - #y Lys Pro Gly Tyr Pro      1               5   - #                10  - #                15               - - Trp Pro                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - Lys Ala Arg Arg Ser Glu Gly Arg Ser Trp Al - #a Gln Pro Gly Tyr Pro      1               5   - #                10  - #                15               - - Trp Pro                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - Lys Ala Arg Arg Ser Glu Gly Arg Ser Trp Al - #a Gln Pro Gly Phe Pro      1               5   - #                10  - #                15               - - Trp Pro                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - Ser Thr Gly Lys Ser Trp Gly Lys                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - Phe Ala Gln Ala Leu Pro Val Trp Ala Arg Pr - #o Asp Tyr Asn Pro Pro      1               5   - #                10  - #                15               - - Leu Val Glu Thr Trp Lys Lys Pro Asp Tyr Gl - #u Pro Pro Val Val His                  20      - #            25      - #            30                   - - Gly                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - Phe Pro Pro Ala Leu Pro Ile Trp Ala Arg Pr - #o Asp Tyr Asn Pro Pro      1               5   - #                10  - #                15               - - Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Va - #l Pro Pro Val Val His                  20      - #            25      - #            30                   - - Gly Phe Pro Pro Ala Leu Pro Ile Trp Ala Ar - #g Pro Asp Tyr Asn Pro              35          - #        40          - #        45                       - - Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Ty - #r Val Pro Pro Val Val          50              - #    55              - #    60                           - - His Gly                                                                  65                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - Phe Pro Pro Ala Leu Pro Ala Trp Ala Arg Pr - #o Asp Tyr Asn Pro Pro      1               5   - #                10  - #                15               - - Leu Val Glu Ser Trp Lys Arg Pro Asp Tyr Gl - #n Pro Ala Thr Val Ala                  20      - #            25      - #            30                   - - Gly                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - Phe Pro Pro Ala Leu Pro Pro Trp Ala Arg Pr - #o Asp Tyr Asn Pro Val      1               5   - #                10  - #                15               - - Leu Ile Glu Thr Trp Lys Arg Pro Gly Tyr Gl - #u Pro Pro Thr Val Leu                  20      - #            25      - #            30                   - - Gly                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - Phe Ala Gln Ala Leu Pro Val Trp                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - Phe Pro Pro Ala Leu Pro Ala Trp Ala Arg Pr - #o Asp Tyr Asn Pro Pro      1               5   - #                10  - #                15               - - Leu Leu Glu Ser Trp Lys Arg Pro Asp Tyr Gl - #n Pro Ala Thr Val Ala                  20      - #            25      - #            30                   - - Gly                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - Pro Asp Tyr Glu Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Pro Asp Tyr Val Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - Pro Gly Tyr Glu Pro Pro Thr Val Leu Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - Ser Gln His Leu Pro Tyr                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - Ala Ser Arg Ala Ala Leu                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - Ala Ser Lys Ala Ala Leu                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - Pro Asp Tyr Arg Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - Pro Asp Tyr Arg Pro Pro Val Val His Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - Pro Asp Tyr Gln Pro Ala Thr Val Ala Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - Phe Ala Gln Ala Leu Pro Val Trp                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - Phe Pro Pro Gln Ala Leu Pro Pro Trp                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - Ser Thr Gly Lys Ser Trp Gly Lys                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - Ser Glu Gly Arg Ser Trp Ala Gln                                          1               5                                                            __________________________________________________________________________

We claim:
 1. A method for detecting one or more types of a hepatitis Cvirus comprising the steps of:(a) providing a biological sample from anindividual suspected of being infected by a hepatitis C virus, (b)contacting the sample with a composition comprising a first reagent saidfirst reagent consisting essentially of a combination of hepatitis Cvirus type specific epitopes specific for a first type of hepatitis Cvirus, wherein said epitopes are from the core, NS4 or NS5 regions ofthe hepaitis C virus and at least one epitopes is derived from the aminoacid sequence spanning amino acids 1689 to 1718 of hepatitis C-virus-1or a homologous region thereof of other hepatitis C virus types, andwherein said contacting is carried out under conditions which allow theformation of first epitope-antibody complexes; and (c) assaying for thepresence of said first epitope-antibody complexes in the sample, whereinsaid assaying performed by a competition assay, a sandwich assay, animmunofluoresence assay, a chemilumenscence assay (CLIA), aradio-immnuoassay, or an enzyme-linked immunosorbent assay.
 2. Themethod of claim 1 further comprising the steps of:(d) contacting thesample with a second reagent comprising a further type specific epitopespecific for a second type of hepatitis C virus, wherein said furtherepitope is selected from the core, NS4, or NS5 region of the hepatitis Cvirus and is selected from a region different from the epitopes in thefirst reagent, and further wherein contacting is carried out underconditions which allow the formation of a second epitope-antibodycomplex; and (c) assaying for the presence of a second epitope-antibodycomplex in the sample.
 3. A method for detecting one or more types of ahepatitis C virus comprising the steps of:(a) providing a biologicalsample suspected of containing HCV antigens; (b) contacting the samplewith a first reagent, said first reagent comprising a combination ofantibodies specific for at least two different type specific epitopesfrom the core, NS4, or NS5 region of a first type hepatitis C virus,wherein at least one antibody in the first reagent is specific for anepitope derived from the aimino acid sequence spanning amino acids 1689to 1718 of hepatitis C virus-1 or a homologous region thereof from otherhepatitis C virus types and wherein said contacting is carried out underconditions which allow the formation of first antigen-antibodycomplexes; (c) assaying for the presence of said first antigen-antibodycomplexes in the sample as an indication of the presence of one or moretypes of hepatitis C virus, wherein said assaying is performed by acompetition assay, a sandwich assay, an immunofluoresence assay, achemilumenscence assay (CLIA), a radio-immunoassay, or an enzyme-linkedimmunosorbent assay; (d) contacting the sample with a second reagentcomprising an antibody specific for a further type specific epitopederived from a second type of hepatitis C virus, wherein said furtherepitope is selected from the core, NS4, or NS5 region of the hepatitis Cvirus, wherein the antibody in the second reagent is specific for anepitope derived from the amino acid sequence spanning amino acids 67 to84 of hepatitis C virus-1 or a homologous region thereof from otherhepatitis C virus types and wherein said contacting is carried out underconditions which allow the formation of a further antigen-antibodycomplex; and (e) assaying for the presence of said furtherantigen-antibody complex in the sample, wherein said assaying isperformed by a competition assay, a sandwich assay, an immunofluoresenceassay, a chemilumenscence assay (CLIA), a radio-immunoassay, or anenzyme-linked immunosorbent assay.
 4. A method for typing one or moretypes of a hepatitis C virus comprising the steps of:(a) providing abiological sample suspected of containing hepatitis C virus antigens;(b) contacting the sample with a first reagent, said first reagentcomprising a combination of antibodies specific for at least twodifferent type specific epitopes from a first type of hepatitis C virus,wherein at least one antibody in the combination is specific for anepitope from the NS4 region of the hepatitis C virus, wherein the NS-4epitope is derived from the amino acid sequence spanning amino acids1689 to 1718 of hepatitis C virus-1 or a homologous region thereof fromother hepatitis C virus types and wherein said contacting is carried outunder conditions which allow formation of first antigen-antibodycomplexes; (c) assying for the presence of antigen-antibody complexes inthe sample as an indication of the presence of one or more types ofhepatitis C virus, wherein said assaying is performed by a competitionassay, a sandwich assay, an immunofluoresence assay, a chemilumenscenceassay (CLIA), a radio-immunoassay, or an enzyme-linked immunosorbentassay; (d) contacting the sample with a second reagent, said secondreagent comprising an antibody specific for a second type specificepitope from the core region of a second type of hepatitis C virus,wherein said second epitope is derived from the amino acid sequencespanning amino acids 67 to 84 of hepatitis C virus-1 or a homologousregion thereof from other hepatitis C virus types and wherein saidcontacting is carried out under conditions which allow the formation ofa second antigen-antibody complex; and (e) assaying for the presence ofa second antigen-antibody complex in the sample as an indication of thepresence of said second hepatitis C virus type in the sample, whereinsaid assaying is performed by a competition assay, a sandwich assay, animmunofluoresence assay, a chemilumenscence assay (CLIA), aradio-immunoassay, or an enzyme-linked immunosorbent assay.
 5. Apolypeptide having an amino acid sequence corresponding to a typespecific epitope within the NS4 region of the hepatitis C virus, whereinsaid epitope is derived from the amino acid sequence spanning aminoacids 1689 to 1718 of hepatitis C virus-1 or a homologous region thereoffrom other hepatitis C virus types, and has a sequence selected from thegroup consisting of CASHLPY (SEQ ID NO:7), CASRAAL (SEQ ID NO:8),CASKAAL (SEQ ID NO:23), ASRAAL (SEQ ID NO:67), and ASKAAL (SEQ IDNO:68).